5-hydroxytryptamine (5-HT) receptors are widely distributed in the central and peripheral nervous systems, regulating and controlling various physiologic functions. 5-HT and its analogues can play both inhibitory or promoting roles in the pain signal conduction via interacting with various subtypes of 5-HT receptor at different locations. In the central nervous system, the descending serotonergic neurons exert analgesic effect by activating the inhibitory interneurons, and the analgesic efficiency depends on the quantity and subtype of the 5-HT receptors. In periphery, 5-HT receptors are involved in pain signal conduction and have a close relationship with hyperalgesia. In this review, we focused on the different subtypes of 5-HT receptors locating in the central and peripheral nervous systems, and summarized the regulation mechanisms of 5-HT receptors in the pain signal, hoping to provide a theoretical basis and new target for the therapy and recovery of acute and chronic pain.